ES2733134T3 - Compositions comprising eicosapentaenoic acid suitable for high purification - Google Patents

Abstract

A heterotrophically cultured diatom that comprises eicosapentaenoic acid and arachidonic acid in a ratio of at least 17: 1, where eicosapentaenoic acid forms at least 1.5% w / w of the dry weight of the diatom and diatom is Nitzschia laevis.

Description

DESCRIPTION

Compositions comprising eicosapentaenoic acid suitable for high purification

Field of the Invention

The field of this invention relates to compositions containing eicosapentaenoic acid that are suitable for concentration and purification for pharmaceutical and industrial use, and methods and procedures for the production of such compositions.

Background of the invention

Pharmaceuticals that contain very long chain omega-3 fatty acids (AG n-3 CML) are currently used to treat hundreds of thousands of patients, and potentially will be used soon to treat millions of patients who are or are at risk of developing cardiovascular disease. In addition, the use of these substances in pharmaceutical products to treat other disorders is being considered.

AG n-3 CML cannot be chemically synthesized de novo economically, therefore, it must be extracted from a biological source. With such sources, most of the n-3 CML AGs will be found in cellular structures such as membranes or lipid bodies and, therefore, will be associated with other molecules that may not be desired in a pharmaceutical product. In these cases, AG n-3 CMLs must be extracted and purified to some extent before use.

Pharmaceutical manufacturers currently rely on fish as a source of AG n-3 CML for the production of pharmacological substances. However, the exclusive dependence on fish oil for such purposes entails a number of serious risks for pharmaceutical manufacturers and pharmaceutical companies, as well as for patients receiving such medications. Such risks include, but are not limited to, those associated with a possible shortage of supplies, which can be financially devastating for pharmaceutical companies, as well as negatively affecting patients who depend on medications for their well-being.

Eicosapentaenoic acid (EPA) is an n-3 CML AG used as an active metabolite in pharmacological substances. To allow patients to tolerate therapeutics well and achieve the levels of bioavailability required for the drug to produce the desired effects, it is desirable that EPA be administered in a highly purified form.

The production of pharmaceutical products or other therapeutic agents from fish oil is complex and there is a possibility that the composition of the final product is altered due to the variability inherent in the method of capture and / or breeding and production of fish oil . There are also widespread concerns about the long-term sustainability of wild fish stocks and aquaculture.

Therefore, there is a great need for alternative sources of EPA to fish oil that are susceptible to a subsequent degree of EPA purification desirable for use in the production of pharmaceuticals or other therapeutic agents.

Wen & Chen describe the production of EPA in the Nitzschia laevis diatom ( Appl. Picrobiol Biotechnol (2001) 57: 316-322) and in other microalgae ( Biotechnology Advances (2003) 21: 273-294). Renaud et al. describe microalgae for use in tropical aquaculture and its chemical composition, which includes EPA. U.S. Patent Document

5 340 594 describes food products that have a high concentration of highly unsaturated omega-3 fatty acids.

Current industrial methods for the production of high purity EPA often perform a concentration stage (in which the short chain fatty acids and saturated fatty acids are removed from the mixture) followed by a purification stage (in which compounds with physiochemical and structural similarity to EPA are removed).

The first method is often unable to separate EPA from arachidonic acid (ARA) and other fatty acids that comprise 20 carbons or more and that contain at least one double bond (fatty acids that concentrate simultaneously), therefore, the development of sources of EPA other than fish oil in which the content of these fatty acids is minimized is highly desirable.

Purification methods experience difficulties in separating molecules that are physiochemically and structurally similar to EPA. The most common molecular species naturally found most similar to EPA is another fatty acid, ARA. The development of EPA sources other than fish oil that are substantially free of such molecules is, therefore, highly desirable.

Such sources should also be ideally free of fatty acids that are not found in appreciable concentrations, or at all, in the diet of common human populations, since their biological activity may not be well characterized, which makes them unsuitable for pharmaceutical use. . This is especially true when said fatty acids have sufficient structural or physiochemical similarity with the EPA that would be difficult to purify from the EPA.

EPA is synthesized as part of a dual parallel biosynthetic pathway that produces omega-3 and omega-6 fatty acids (for example, see Damude and Kinney ( Lipids 42: 179-185, 2007)). Many of the enzymes are shared between the pathways and will act on the omega-3 or omega-6 form of a fatty acid, so the products of the omega-6 pathway are commonly found along with those of the omega-3 pathway. In particular, the ARA is commonly found together with the EPA. Each stage of the synthetic route is catalyzed by separate enzymes and it is not uncommon for the intermediate products of the route to accumulate, these will include fatty acids with a smaller number of double bonds but the same number of carbon molecules. ARA and EPA can also be substrates for greater elongation and desaturation in fatty acids such as docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Therefore, it is not surprising that EPA-containing organisms generally also contain significant amounts of ARA and fatty acids that are concentrated simultaneously.

Therefore, there is a need in the art to provide EPA in a form that is susceptible to high-grade purification for therapeutic use and in abundance. In particular, there is a need to provide EPA in a form that is separable from other fatty acids that are physiochemically and structurally similar to EPA (for example, ARA).

Compendium of the invention

In a first aspect, the invention provides a heterotrophically cultured diatom that comprises eicosapentaenoic acid (EPA) and arachidonic acid (ARA) in a ratio of at least 17: 1 where the EPA forms at least 1.5% w / w of the weight Dry from the diatom and diatom is Nitzschia laevis.

Preferably there is no juniperonic acid (JPA) and / or sciadonic acid in the fatty acids that are concentrated simultaneously.

In a second aspect, the invention provides a method for producing a diatom that comprises eicosapentaenoic acid (EPA) and arachidonic acid (ARA) in a ratio of at least 17: 1 and at least 1.5% w / w of the dry weight of the diatom. es e Pa, which includes the stages of:

to. culturing diatoms in heterotrophic cultures that contain a nutrient solution comprising:

(i) an organic carbon source at an initial concentration of at least 0.5 M carbon (ii) a mole ratio of organic carbon to nitrogen of not more than 30: 1 (mol C: mol N) and;

(iii) a non-limiting source of silicate that does not fall below 150 | jM; Y

b. recover the diatom of the heterotrophic culture.

Preferably, the method further comprises the step of extracting fatty acids from said biomass to produce a fatty acid composition.

Preferably, the method further comprises the step of enriching the fatty acid composition in EPA or purifying EPA from the fatty acid composition to produce an enriched or purified fatty acid composition.

Preferably the diatom is Nitzschia laevis.

Preferably, diatoms are grown in heterotrophic culture in a fermenter.

Preferably, the culture comprises continuous fermentation.

Preferably the diatoms comprise Nitzschia species .

Preferably, the culture is carried out at a temperature of about 12 ° C to about 35 ° C. Preferably, the culture is carried out at a pH of about 7.0 to about 8.7.

Preferably, said carbon source comprises glucose.

Preferably, said carbon source is selected from glucose, hydrolyzed starch or hydrolyzed whey. Preferably, said nitrogen source is in the form of sodium nitrate or potassium nitrate.

Preferably, said nitrogen source is enriched with an amino acid source.

Preferably, said source of amino acids is selected from corn mash liquor, yeast extract, tryptone, peptones, lysine and glutamate.

Preferably said silicate is in the form of sodium or potassium metasilicate.

Preferably, the nutrient solution comprises phosphorus in the form of phosphate.

Preferably, the mole ratio of organic carbon to phosphorus in the form of phosphate is not greater than 1250: 1 (mol C: mol P).

Preferably, the mole ratio of carbon to silicon in the form of a silicate source is 100: 1 to 850: 1.

Preferably, the nutrient solution comprises phosphorus in the form of phosphate.

Preferably, the mole ratio of carbon to phosphorus in the form of phosphate is 100: 1 to 1250: 1.

Brief description of the drawings

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

Figure 1: shows the relationship between EPA: ARA ratios in biomass and minimum concentrations of sodium metasilicate in the medium in which the biomass is formed.

Detailed description of the invention

Unexpectedly, the present authors have found that it is possible to obtain compositions of heterotrophic microorganisms comprising EPA containing an EPA to ARA ratio of about 11: 1 or more, while at the same time also containing EPA at more than 8 times the level of all the fatty acids that are concentrated simultaneously combined. Such microorganisms can be produced in culture at a yield rate greater than 5 mg of EPA / L / h and with an EPA content greater than 1.5% of the total dry cell weight, which makes them relevant in an industrial process.

Also unexpectedly, the inventors have found that it is possible to obtain heterotrophic diatom compositions comprising EPA containing an EPA to ARA ratio of about 17: 1 or more. Such microorganisms can be produced in culture at a yield rate greater than 5 mg of EPA / L / h and with an EPA content greater than 1.5% of the total dry cell weight, which makes them relevant in an industrial process. A part of the present invention is the recognition that it is not only desirable, for the purpose of concentrating the fatty acid mixture, to control the amounts of fatty acids that are concentrated simultaneously (as defined herein) in relation to the EPA that can accumulate in the heterotrophic culture of microorganisms, but it is also feasible to do so through the control of the growth conditions of these microorganisms.

Another part of the present invention is the recognition that it is not only desirable to control the relative amounts of physiochemically or structurally similar molecules to EPA that can accumulate in the heterotrophic culture of microorganisms for the purposes of EPA purification, but that it is also feasible to do so through the control of the growth conditions of these microorganisms.

Another part of the present invention is the recognition that it is the relative levels of the molecules, rather than the absolute levels, that are physiochemically and structurally similar to the EPAs, that are critical for concentration and purification and that, surprisingly , the maximum production of EPA in biomass does not necessarily equal the maximum production of highly purified EPA.

Accordingly, the invention provides a heterotrophic diatom culture that comprises eicosapentaenoic acid and arachidonic acid in a ratio of at least 17: 1 where the eicosapentaenoic acid forms at least 1.5% w / w of the dry weight of the diatom and Diatom is Nitzschia Laevis.

The invention further provides: a method for producing a diatom that comprises eicosapentaenoic acid and arachidonic acid in a ratio of at least 14: 1 and at least 1.5% w / w of the weight of dry cells such as eicosapentaenoic acid comprising:

to. culturing diatoms in heterotrophic cultures that contain a nutrient solution comprising:

(i) an organic carbon source at an initial concentration of at least 0.5 M carbon (ii) a mole ratio of organic carbon to nitrogen of not more than 30: 1 (mol C: mol N) and;

(iii) a non-limiting source of silicate that does not fall below 150 | jM; and

b. recover the diatom of the heterotrophic culture.

Definitions and Abbreviations

Omega-3 fatty acid is a fatty acid with the first double bond to three carbon atoms of the n-methyl end of the molecule. Omega-3 often shortens to n-3.

Omega-6 fatty acid is a fatty acid with the first double bond to six carbon atoms of the n-methyl end of the molecule. Omega-6 often shortens to n-6.

Fatty acids are described in the form CX: Y, where the number X describes the number of carbon atoms and the number Y describes the number of double bonds in the fatty acid. Where Y is equal to zero, the fatty acid is described as saturated, where Y is greater than zero, the fatty acid is described as unsaturated. The position and type of the double bonds can be specified as, for example, "cis 5, 11, 14" where the numbers reflect the location of the carbon-carbon double bonds, counting from the carboxylic acid end of the molecule. It is understood that a term such as C20: 5 includes both free fatty acid and esterified forms of the fatty acid with the number of carbon atoms and double bonds that refer only to the fatty acid portion of the ester.

EPA, C20: 5 n-3, eicosapentaenoic acid, is an omega-3 fatty acid with twenty carbon atoms and five double bonds.

ARA, C20: 4 n-6, arachidonic acid, is an omega-6 fatty acid with twenty carbon atoms and four double bonds.

Fatty acids that concentrate simultaneously are defined as fatty acids, excluding EPA and ARA, which comprise 20 carbons or more and contain at least one double bond. These include, but are not limited to the various forms of C20: 1, C20: 2, C20: 3, C20: 4, C22: 5, C22: 6 and C24: 1.

DHA, C22: 6 n-3, docosahexaenoic acid, is an omega-3 fatty acid with twenty-two carbon atoms and six double bonds.

DPA, C22: 5 n-3, docosapentaenoic acid, is an omega-3 fatty acid with twenty-two carbon atoms and five double bonds.

DGLA (C20: 3 n-6), dihomo-gamma-linoleic acid is an omega-6 fatty acid with twenty carbon atoms with three double bonds.

ETAn-3 (C20: 4 n-3), eicosatetraenoic acid is an omega-3 fatty acid with twenty carbon atoms and four double bonds.

TFA (total fatty acids) means the sum of all fatty acids in a composition or mixture.

DCW (dry cell weight) means the weight of a biomass once all the water has been removed.

Heterotrophic culture means a culture of organisms for which at least 90% of the energy supply for the crop is obtained from the nutrients supplied that are usually a form or forms of organic carbon (e.g., glucose, acetate) . Therefore, a maximum of 10% of the energy supply is obtained from the light energy. Preferably, less than 5% or less than 1% of the energy supply is obtained from the light energy. More preferably, the entire energy supply comes from the nutrients supplied.

Photoautotrophic culture means a culture of organisms for which the only source of energy is light.

Mixotrophic culture means a culture of organisms for which the energy source is a mixture of 10% or more of light energy and less than 90% of nutrients derived from nutrients.

The limitation of nutrients means that the absence or low level of the nutrient in question causes the body to grow more slowly than it would if the nutrient were present at higher levels. Therefore, the non-limiting provision of nutrients means that factors other than the nutrient in question are limiting in the growth of the organism and the provision of greater amounts of the nutrient in question would not have the effect of increasing growth. Therefore, the nutrient is present in sufficient quantities so that it is not depleted by its incorporation into the biomass, so that the free concentration in the nutrient solution does not fall to zero. Compositions of the invention

Genetically modified microorganisms are potentially capable of accumulating significant levels of EPA when produced in heterotrophic cultures. Damude et al. in US20060115881 provide compositions comprising EPA produced in genetically modified yeast strains. However, they contain relatively low EPA ratios with respect to other molecules that are physiochemically and structurally similar to EPA, which makes them poorly suited for concentration and purification. The authors teach nothing about the relevance of fatty acids that concentrate simultaneously on the production of purified EPA and do not suggest how this problem could be addressed.

Xue et al. in the US patent document 2009/0093543 provide compositions comprising EPA produced in genetically modified yeast strains. One of these compositions is now commercially available and is currently marketed as Futurebiotics Newharvest OMEGA-3. The compositions described contain particularly high amounts of EPA (more than 50% of the total fatty acids in some cases) and low amounts of ARA, but contain significant amounts of C20 fatty acids that are simultaneously concentrated that make them unsuitable for the present purpose. . In addition, the authors describe the presence of juniperonic acid (C20: 4 cis 5, 11, 14, 17) in the fatty acid profile of at least one of their genetically modified yeast strains. This fatty acid is not only physiochemically and structurally similar to EPA, which makes it particularly difficult to separate, but also is not normally found in the human diet, which makes it particularly problematic as a contaminant in any EPA product. purified for use in humans. The authors do not teach the importance of reducing the proportions of fatty acids that simultaneously contaminate the production of purified EPA or how such compositions could be achieved.

In US patent documents 5683898 and 5798259, Yazawa et al. They provide transgenic E. coli cultures with EPA at around 1.5% of total fatty acids. The authors do not specify the presence or absence of other fatty acids in their material and do not discuss the purification of EPA from their material. The authors teach nothing about the relevance of fatty acids that concentrate simultaneously on the production of purified EPA and do not suggest how this problem could be addressed.

Nor Damude et al., Xue et al., Yazawa et al. nor has any other author provided compositions comprising EPA produced in transgenic microorganisms that at the same time contain sufficiently high ratios of EPA with respect to other physiochemically or structurally similar fatty acids to make them suitable for concentration and purification.

In addition, neither Damude et al., Xue et al., Yazawa et al. nor has any other author taught that cultured microorganisms should comprise prescribed ranges for EPA's relationships with respect to other physiochemically or structurally similar molecules to those taught herein, nor do they specifically teach how the elimination of such molecules should be achieved. .

Certain prokaryotic microorganisms, which include but are not limited to certain strains of non-genetically modified (non-GE) marine bacteria have an inherent ability to produce de novo Pa and are capable of accumulating amounts of EPA in culture without resorting to genetic modifications. For example, Yazawa et al. ( J. Biochem.

103: 5-7 1988) provide compositions comprising EPA of the SCRC-8132 non-GE bacteria. The authors state that "surprisingly, other polyunsaturated fatty acids, such as arachidonic (C20: 4) ....... were not detected at all [in the cultured bacteria]" and "this unique composition of fatty acids makes it easy to isolate the EPA from a culture of this bacterial strain, "but does not comment on other C20 fatty acids and their role in concentration. In addition, the authors do not teach how high levels of EPA and low levels of ARA can be achieved in the culture of a eukaryotic organism. Bowman et al. ( Int J Syst Bacteriol 47: 1040-1047, 1997) provide compositions comprising prokaryotic microorganisms with high levels of EPA as a proportion of total fatty acids. The authors do not record C20 fatty acids other than EPA and ARA, and in some cases they appear not to have measured ARA, so they do not reveal our composition. The authors also do not teach the relevance of such compositions for concentration and purification, nor do they suggest how such compositions could be obtained in the culture of a eukaryotic organism.

None of these prokaryotic sources have been commercialized due to low levels of performance. The maximum rate of return reported by Yazawa et al. for example, it is 1.1 mg of EPA / L / h but EPA forms less than 0.5% of the dry cell weight. Non-GE prokaryotes cannot compete as a source of EPA with eukaryotic microorganisms due to the relatively low dry weight of the EPA and, therefore, are poorly suited for the current purpose. Since prokaryotic and eukaryotic organisms are fundamentally different, the teachings of one cannot be applied directly to the other, so there can be no reasonable expectation that the observations of Yazawa et al., Or Bowman et al. nor from any other author about the EPA content of naturally occurring prokaryotic organisms can be used to further develop the production of eukaryotic EPA.

Several authors provide compositions comprising EPA as a component of a microorganism produced in cultures of naturally occurring eukaryotic microorganisms that use light as their main source of energy for growth. If light is the only source of energy, these crops are considered photosynthetic or photoautotrophic. Organic carbon can be added to a photosynthetic culture to convert it into myxotrophic. These crops generally occur in open ponds or tracks, or in photobioreactors supplied internally with large amounts of artificial light and / or compounds of transparent material with high surface to volume ratios to allow light to pass to the crop. Low returns, high capital costs and / or technical difficulties associated with such production and the consequent costs of producing EPA in this way make them unsuitable for the current purpose.

For example, Vazhappilly and Chen ( JAOCS 75: 393-397, 1998) provide compositions comprising EPA in several species of microalgae grown under photoautotrophic conditions and the best of them only showed average growth rates of around 256 mg of cells per day. , and many of them show much lower. The authors point out that "the high accumulation of arachidonic acid (ARA, C20: 4n-6) with EPA or DHA is disadvantageous because ARA can have detrimental effects on health and problems in the recovery of EPA", but they do not suggest means to reduce ARA content in heterotrophic cultures. In addition, the authors state that "the culture of ARA was relatively low in the 20 microalgae except Porphyridium] cruentum." Since many of the mentioned species have very similar amounts of the two fatty acids and two even have more ARA than EPA, this suggests that the authors consider that the absolute amount of ARA is important instead of the ratio of EPA to ARA. This teaches far from our invention, since we teach that it is the ratio of EPA to ARA that is important rather than the absolute amount of any of the fatty acids.

Certain non-GE eukaryotic microorganisms, which include but are not limited to microalgae, fungi and yeasts, can grow heterotrophically and can accumulate significant levels of EPA when produced in heterotrophic cultures. These have the advantage that they can be produced indoors in conventional steam sterilizable reactors with relatively low surface to volume ratios compared to photobioreactors.

Yongmanitchai and Ward ( Process Biochemistry 24: 117-125, 1989) provide a list of microorganisms that produce EPA and DHA. The authors do not distinguish between organisms that can be cultured in a heterotrophic way or should be cultivated photosynthetically, so it is impossible to anticipate from this reference if these organisms are suitable for the industrial production of EPA. It is also not possible to determine if the fatty acid composition data described comes from heterotrophic cultures. In no case, the data provided on the fatty acid content of the organisms allow the complete identification of the EPA content in relation to the ARA and other fatty acids that are concentrated simultaneously, so as to describe the composition of this invention. The authors discuss the production of high purity EPA, but do not discuss the effect that other polyunsaturated fatty acids have on the ease with which EPA can be purified. The reference does not suggest the composition described herein, so that said composition is relevant for concentration and purification, nor that there is an expectation that one can succeed in obtaining said composition.

Barclay in US patent documents 5130242 and 5908622 discloses the isolation, heterotrophic growth and fatty acid profiles of several strains of algae. The simultaneous presence of EPA and the absence of ARAs are reported for several of these (growth under standard selection conditions), while an additional number has low levels of ARA compared to EPA. Most of these have EPA: DHA ratios of less than 8, which makes them unsuitable for current purposes.

The BRBG strain is disclosed in Table 3 of US Pat. 5130242 as having no AA (ARA) and an EPA to DHA ratio of 12 and in Table 4 that has an EPA to DHA ratio of 10.9. The same data is used in US Pat. 5908622.

However, dihomo-gamma-linoleic acid (DGLA) or eicosatetraenoic acid (ETA) is not disclosed, so the author clearly does not recognize the importance of relative levels of other long chain fatty acids of 20 carbons or more for the purpose of concentration and purification. Given the relatively low amounts of EPA in the fatty acid profile, even small amounts of these or other C20 compounds could have a significant effect on the ratio of EPA to simultaneously concentrated fatty acids. No productivity data is provided for the growth of these strains.

The author considers the purification of omega-3 fatty acids as a group, but does not consider the purification of individual fatty acids, so he does not teach the relevance of the composition for the concentration and purification of individual fatty acids.

While none of the omega-6 fatty acids, including ARA, are disadvantageous for dietary purposes, the author states that "strains that have less than 1% (as% of total fatty acids) of HUFA (acids can also be isolated) highly unsaturated fatty acids), for its acronym in English, C20: 4n-6 and C22: 5n-6 undesirable for some applications, "indicating that this author considers the total amount instead of the relative amount of ARA to be important. This teaches far from the present invention that it is the ratio of EPA to ARA that is important rather than the absolute amount of any of the fatty acids.

Several authors provide compositions comprising EPA produced in diatoms. For example, Tan and Johns ( Journal of Applied Phycology 8: 59-64, 1996) disclose the heterotrophic growth of several diatoms, but do not record or mention the ARA content of the cells or the content of other fatty acids that are simultaneously concentrate other than C20: 3. The reference does not suggest the composition described herein, therefore that said composition is relevant for concentration and purification, nor that there is an expectation that one can succeed in obtaining said composition.

Kitano et al. ( Journal of Applied Phycology 9: 559-563, 1997) disclose the heterotrophic growth of the diatomea Navícula saprophila, but the ratio of EPA to C20: 4 obtained is less than 10. The content of other long-chain fatty acids of 20 carbons or more is not described, nor is any consideration given to purification. The reference does not suggest our composition, so such a composition is relevant for concentration and purification, nor is there any expectation that one can succeed in obtaining such a composition.

In US patent documents 5244921 and 5567732, Kyle and Gladue describe methods for producing eicosapentaenoic acid from the Nitzschia alba diatom in a heterotrophic culture. In cells produced by their methods, the ratio of EPA to C20: 4 is only 4: 1. The ratio of EPA to other fatty acids that are concentrated simultaneously is not considered nor is the probable effect on purification. The reference does not suggest our composition, so such a composition is relevant for concentration and purification, nor that there is an expectation that one can succeed in obtaining such a composition.

Chu et al. ( Journal of Applied Phycology 8: 389-396, 1996) disclose the heterotrophic culture of the Nitzschia inconspicuous diatom to acetate and glucose. The authors do not describe the long chain fatty acid content of 20 carbons or more, other than EPA and ARA. The ratio of these two fatty acids is, in any case, less than 2 under heterotrophic conditions. The reference does not suggest the composition described herein, so that a composition is relevant for concentration and purification, nor that there is an expectation that one can succeed in obtaining such a composition.

Wen and Chen in several articles ( Biotechnology Letters 22: 727-733, 2000; Journal of Industrial Microbiology & Biotechnology 25: 218-224, 2000; Enzyme and Microbial Technology 29: 341-347, 2001; Biotechnol Bioeng 75: 159 169, 2001 .; Biotechnol. Prog. 18: 21-28, 2002; Process Biochemistry 37: 1447-1453, 2002; Process Biochemistry 38: 523-529, 2002) have described the optimization of heterotrophic growth conditions for the Nitzschia laevis diatom . The authors work to maximize the production of EPA but have not given any indication that the ratio of EPA to ARA is important or that the optimization of the relationship is considered. Similarly, the relative levels of EPA with respect to fatty acids that are concentrated simultaneously, nor the concentration or purification are not taken into account. The references do not show the composition described herein, nor do they suggest that this composition is relevant for concentration and purification.

In Pahl et al. ( J Bioscience and Bioeng 109: 235-239, 2010) The authors report the heterotrophic growth of the Cyclotella cryptica diatom with the purpose of producing feed for use in the aquaculture industry. The authors do not consider the possibility of producing purified EPA or the effect that relative levels of EPA with respect to other long-chain fatty acids would have on concentration or purification. In particular, the amounts of ARA are not even disclosed in the document. The authors use a medium rich in sources of nitrogen, phosphates and silicates, but with relatively low amounts of carbon. As a result, the EPA content of the cells remains below 1.5% of the dry cell weight. The reference does not suggest the composition described herein, so that a composition is relevant for concentration and purification, nor that there is an expectation that one can succeed in obtaining such a composition.

Griffiths and Geiringer in WO2008004900 provide compositions comprising EPA with low levels of molecules with structural and physicochemical properties similar to EPA obtained from diatoms. These authors describe the general convenience of compositions comprising EPA with low levels of certain fatty acids for pharmacotherapeutic use. In particular, ARA and other omega-3 and omega-6 fatty acids are classified which "can decrease the desired effect through actions that can be antagonistic, competitive, block, reverse, mediate, create synergies or otherwise alter the effect. EPA's desired health benefits "as unwanted. The authors also describe the general convenience of compositions comprising EPA with low levels of the unwanted fatty acids mentioned above for the purpose of purification for subsequent pharmacotherapeutic use. They do not consider other C20 fatty acids that have no effect on the beneficial health effect of EPA, but may affect the degree to which it can be concentrated or purified.

While the authors teach the convenience of EPA compositions with low levels of ARA for therapeutic purposes, they fail to suggest the compositions of the present invention because their compositions require a first stage of biomass isolation, and their compositions do not take into account all fatty acids that are concentrated simultaneously, simply those that have effects on the biological activity of EPA. Furthermore, it is not taught how the compositions of the present invention could be obtained or the industrial use of doing so.

In addition, neither Griffiths nor Geiringer, nor any other author have taught that the prescribed ranges for EPA to ARA ratios or the fatty acid molecules that are concentrated simultaneously as taught herein are important in the heterotrophic culture of microorganisms by which result in a composition susceptible to a high degree of concentration and after purification.

While heterotrophic cultures of microorganisms, and in particular eukaryotic microalgae of natural origin, can act as an alternative source of omega-3 fatty acids for fish through the accumulation of large amounts of EPA in the culture, it may seem surprising that relatively little or no systematic effort has been devoted to the development of such EPA sources that are at the same time sufficiently free of physiochemically or structurally similar molecules to the EPA to allow a high degree of concentration and purification.

In a first broad aspect, the invention provides a heterotrophic growth diatom, Nitzschia laevis, in which the ratio of EPA to ARA is at least 17: 1 and EPA is at least 1.5% of the dry cell weight, where the ratios They are on a weight basis.

The invention further provides: a method for producing a diatom that comprises eicosapentaenoic acid and arachidonic acid in a ratio of at least 14: 1 and at least 1.5% w / w of the weight of dry cells such as eicosapentaenoic acid comprising:

to. culturing diatoms in heterotrophic cultures that contain a nutrient solution comprising:

(i) an organic carbon source at an initial concentration of at least 0.5 M carbon

(ii) a mole ratio of organic carbon to nitrogen of not more than 30: 1 (mol C: mol N) and;

(iii) a non-limiting source of silicate that does not fall below 150 pM; Y

b. recover the diatom of the heterotrophic culture.

Preferably, the composition comprises an EPA to ARA ratio of at least 15: 1.

Preferably, the composition comprises an EPA ratio to fatty acids that are simultaneously concentrated at least 9: 1, more preferably the ratio is at least 10: 1, more preferably at least 11: 1 and even more preferably at least minus 12: 1.

Preferably, less than 1% w / w of the total fatty acids is juniperonic acid (which is a fatty acid that is concentrated simultaneously). More preferably, the composition comprises less than 0.5% w / w juniperonic acid. Most preferably, juniperonic acid is not present in the composition.

Preferably, less than 1% w / w of the total fatty acids is sciadonic acid (which is a fatty acid that is concentrated simultaneously). More preferably, the composition comprises less than 0.5% w / w of sciadonic acid. Most preferably, sciadonic acid is not present in the composition.

Preferably, the heterotrophic cultured microorganisms are diatoms of the genus Nitzschia, even more preferably the microorganisms are comprised of the marine unicellular diatomome known as Nitzschia laevis.

Preferably, the biomass composition comprises a proportion of its total dry weight as fatty acids; the proportion that is in the range of 5 to 80%, a proportion of which will be EPA; the proportion of EPA (dry weight of fatty acids) in the range of 2 to 80%, more preferably the proportion is more than 10%, more preferably more than 20% and even more preferably more than 30% . Typically, the proportion (in dry weight) of total fatty acids that is EPA is in the range of 10 to 80%, 20 to 80% or 30 to 80%.

Typically, microbial biomass is produced from a microbial culture by collecting microbes from the heterotrophic culture medium, optionally, heat or destruction of cells (for example, to denature endogenous enzymes) and cell formation in a biomass (e.g., a biomass cake). The biomass is optionally dried to reduce or remove water. The biomass formation of the invention does not include the extraction or purification steps to recover fatty acids or materials other than water. However, the EPA: ARA ratio of the biomass of the invention serves to facilitate the subsequent stages of extraction and purification used to enrich the EPA for therapeutic purposes. The microbial biomass of the invention can be subjected to one or more extraction steps to extract fatty acids from the biomass to produce a fatty acid composition. Suitable extraction techniques are well known in the art. For example, the biomass can be extracted with a non-selective lipid solvent (eg, dimethyl ether or quasi-critical ethanol) and recovered from the solvent as a residue.

After extraction, the level of EPA in said fatty acid composition is 5 to 90%, 5 to 75% or 5 to 50% by weight of the fatty acids in the composition. The ratio of EPA to ARA can be maintained at substantially the same level as before extraction, preferably at least 14: 1. The ratio of EPA to total fatty acids that are concentrated simultaneously can be retained substantially at the same level as before extraction, preferably at least about 8: 1.

After extraction, the additional step of enriching the fatty acid composition in EPA or purifying EPA from the fatty acid composition can be performed using techniques well known in the art. For example, the extracted material can be treated with acids, alkalis and / or enzymes in the presence of alcohol or water for form mixtures of alkyl esters of fatty acids or fatty acids. The mixtures can then be concentrated and further purified to achieve a required purity standard. The ratio of EPA to ARA in the composition can be maintained substantially at the same level as before enrichment and / or purification, preferably at least 14: 1. The ratio of EPA to total fatty acids that are simultaneously concentrated in the composition can also be retained substantially at the same level as before enrichment and / or purification, preferably at least about 8: 1. More preferably, the ratios of EPA to ARA and EPA to total fatty acids that are concentrated simultaneously will increase after the enrichment and / or purification stage. However, given the relative difficulty and cost of separating EPA from ARA and fatty acids that are concentrated simultaneously, there is an advantage in having relatively high ratios of EPA to ARA and EPA to fatty acids that simultaneously concentrate on the composition before the enrichment and / or purification stage.

After enrichment or purification, the level of EPA in said fatty acid composition is at least 30%, or at least 50%, or is a high purity EPA composition of at least 60%, at least about 70%, at least 80%, at least 90%, at least 95% or at least 97% by weight of the fatty acids in the composition. The composition of high purity EPA for the purposes of this specification can be defined accordingly. Such a high purity EPA composition that forms another aspect of the invention.

The heterotrophic growth diatom of the invention or produced by the method of the invention can be used in the manufacture of a product for human or animal consumption. Such a product could be formulated by one skilled in the art using known techniques, and may include, for example, fillers, vehicles, buffers, stabilizers and preservatives. It can take the form of, for example, tablets, capsules, liquids, solutions, and can also be combined with other food products such as breads and cereals, spreads and dairy products.

It can be mixed with other fatty acids or alkyl esters of fatty acids.

The heterotrophic growth diatom of the invention or produced by the method of the invention can be used in the manufacture of a medicament for the treatment of a person affected by certain medical conditions or disorders that include, but are not limited to those selected from the diabetes (type I, and type II), glycemic disorders, hypertension associated with diabetes, cancer, osteoarthritis, autoimmune diseases, rheumatoid arthritis, inflammatory and autoimmune diseases other than arthritis, respiratory diseases, neurological disorders, neurodegenerative disorders (including Huntington's disease, Parkinson's disease, Alzheimer's disease, schizophrenia, major depression, unipolar depression, bipolar depression, obsessive compulsive disorder, borderline personality disorder, postpartum depression, organic brain damage and traumatic brain injury), kidney and tract disorders urinary, cardiovascular disorders ares, cerebrovascular disorders, degenerative diseases of the eyes, psychiatric disorders, reproductive disorders, visceral disorders, muscular disorders, metabolic disorders, prostatic hypertrophy and prostatitis, impotence and male infertility, mastalgia, male pattern baldness, osteoporosis, dermatological disorders, dyslexia and other learning disabilities, cancer cachexia, obesity, ulcerative colitis, Crohn's disease, anorexia nervosa, burns, osteoarthritis, osteoporosis, attention deficit / hyperactivity disorder, and early stages of colorectal cancer, lung and kidney diseases, and associated disorders with abnormal growth and development. Preferably, the medical condition of the disorder is a cardiovascular disorder or a condition related to obesity.

Methods of the invention

Several authors teach methods by which diatoms can be cultured to produce EPA.

For example, Tan and Johns ( Journal of Applied Phycology 8: 59-64, 1996) disclose the heterotrophic growth of several diatoms, but do not record or mention the ARA content of the cells or the fatty acid content with more than 20 carbon atoms other than C20: 3. In addition to examining the modes of photosynthetic and myxotrophic growth, the authors do not attempt any variation in nutrients or comment on the fact that this could affect the composition of fatty acids. These authors do not suggest the method of the present invention since they do not recognize that the relative ratios of nutrients of carbon, nitrogen, silicate and phosphate will have an effect on the composition of fatty acids. Nor do the authors describe that it might be desirable to affect the relative relationships of EPA to ARA in biomass through the nutrient content of growth media.

Kitano et al. ( Journal of Applied Phycology 9: 559-563, 1997) disclose the heterotrophic growth of the Navicula saprophila diatom , but the ratio of EPA to C20: 4 obtained is less than 10. In addition to examining the photosynthetic and myxotrophic growth modes , the authors do not attempt any variation in nutrients or comment on the fact that this affects the composition of fatty acids. These authors do not suggest the method of the present invention since they do not recognize that the relative ratios of nutrients of carbon, nitrogen, silicate and phosphate will have an effect on the composition of fatty acids. Nor do the authors describe that it might be desirable to affect the relative relationships of EPA to ARA in biomass through the nutrient content of growth media.

In US patent documents 5244921 and 5567732, Kyle and Gladue describe methods for producing eicosapentaenoic acid from the Nitzschia alba diatom in a heterotrophic culture. In this, the authors teach far from the present invention, depriving the cells first of nitrogen and then silicate before harvest. The Cells accumulate lipids, but the ratio of EPA to C20: 4 is only 4: 1. The ratio of EPA to other long-chain fatty acids of 20 carbons or more is not considered nor is the probable effect on purification.

Wen and Chen in several articles ( Biotechnology Letters 22: 727-733, 2000; Journal of Industrial Microbiology & Biotechnology 25: 218-224, 2000; Enzyme and Microbial Technology 29: 341-347, 2001; Biotechnol Bioeng 75: 159 169, 2001 .; Biotechnol. Prog. 18: 21-28, 2002; Process Biochemistry 37: 1447-1453, 2002; Process Biochemistry 38: 523-529, 2002) have described the optimization of heterotrophic growth conditions for the Nitzschia laevis diatom . The authors have manipulated nutrient levels in their feeding media to maximize EPA production, but have not attempted to measure any residual nutrient other than glucose, and therefore have not investigated the effects of nitrogen deficiency limitation. , phosphate or silicate of nutrients in the crop itself. The authors have not reached the prescribed intervals for nutrient component ratios as taught herein, so a sufficiently high ratio of EPA to ARA can be obtained.

These authors do not suggest the method of the present invention, since they do not recognize that the limitation due to lack of nitrogen or silicate nutrients has an effect on the relative relationships of EPA to ARA in biomass. Nor do the authors describe that it might be desirable to affect the relative relationships of EPA to ARA in biomass through the nutrient content of the culture media.

In Pahl et al. (J Bioscience and Bioeng 109: 235-239, 2010) The authors report the heterotrophic growth of the Cyclotella cryptica diatom with the purpose of producing feed for use in the aquaculture industry. The authors use a medium rich in nitrogen sources, phosphates and silicates, but with relatively low amounts of carbon, so the prescribed nutrient composition is not achieved as taught herein. The authors do not consider the possibility of producing purified EPA or the effect that relative levels of EPA with respect to ARA would have on concentration or purification; The amounts of Ar a are not even disclosed in the document. The authors point out that one might expect that silicate limitation increases the proportion of fatty acids with a lower degree of unsaturation, but does not consider that changing nutrient ratios in the media can affect the relationship between different highly unsaturated long chain fatty acids. . These authors do not suggest the method of the present invention, since they do not recognize that the relative ratios of nutrients of carbon, nitrogen, silicate and phosphate will have an effect on the relative ratios of EPA to ARA in biomass. Nor do the authors describe that it might be desirable to affect the relative relationships of EPA to ARA in biomass through the nutrient content of the culture media.

Roessler (J Phycol 24: 394-400, 1988) examines the effects of silicon deficiency in photoautotrophic cultures of the Cyclotella cryptica diatom . The author notes that silicon deficiency increases the amount of lipids in cellular matter, but decreases the relative proportion of PUFA and even suggests that if biomass is produced for PUFA production, the growth medium should be packed with nutrients. However, the author does not comment on any change in the relationship between PUFAs that cause silicate deficiency. In addition, the concentration or purification of EPA from the material and the importance of relative levels of EPA with respect to other long-chain fatty acids of 20 carbons or more for this purpose are not taken into account. The author does not suggest the method of the present invention, since his method uses light as the main source of energy for the crop. Nor do they suggest success in methods that do not use light in the main source of energy, since it is not recognized that the relative ratios of nutrients of carbon, nitrogen, silicate and phosphate have an effect on the relative ratios of EPA to ARA in the biomass The author also does not describe that it may be desirable to affect the relative relationships of EPA to ARA in biomass through the nutrient content of growth media.

Taguchi et al. ( J Phycol 23: 260-267, 1987) examine the lipid content of marine diatoms under conditions of silicate deficiency under photoautotrophic conditions. The authors state that "it is essential to establish an N: P: Yes critical relationship so that it can be ensured that the algae use silicate at first compared to nitrate and phosphate. The low remaining concentration of nitrate and phosphate keeps the algae alive to produce more lipids per cell, even when the algae cannot grow anymore due to the depletion of silicate from the medium, "indicating that they believe that silicate deprivation is beneficial and their teaching moves away from this invention. The authors do not describe or consider the fatty acid composition of the cells. The authors do not suggest the method of the present invention, since their method uses light as the main source of energy for the crop. Nor do they suggest success in methods that do not use light in the main source of energy, since the authors teach far from the present invention by depriving silicate cells to increase lipid yield.

Chu et al. ( Journal of Applied Phycology 8: 389-396, 1996) disclose the heterotrophic culture of the Nitzschia inconspicuous diatom on acetate and glucose and also examine the effects of the changing levels of silicon and nitrogen available in the photosynthetic culture. The authors do not comment on the effect that these changes have on the relationship between EPA and ARA, nor is there any consideration of the concentration or purification of EPA from the material and the importance of relative levels of EPA with respect to other chain fatty acids. long of 20 carbons or more for this purpose. In addition, the authors also state that "the lack of nitrogen increased the total fatty acid content of N. inconspicua, and the highest yield of 20: 5 (n-3) occurred when they grew at a low level of NaNO3." Indicating, contrary to the present invention, that they believe that EPA performance is the most important factor in determining growth conditions. The authors do not suggest the method of the present invention since most of their methods use light as the main source of energy for the crop.

Nor do they suggest success in methods that do not use light in the main source of energy, since the authors teach far from the present invention by depriving nitrogen cells to increase lipid yield.

Barclay in US patent documents 5130242 and 5908622 discloses the isolation, heterotrophic growth and fatty acid profiles of several strains of algae (not diatoms). The author teaches that "if a product significantly higher in lipids and omega-3 highly unsaturated fatty acids is desired, the culture can be manipulated to be limited in nutrients, preferably, limited in nitrogen for a suitable time ...", therefore, it teaches directly away from the invention. In addition, the author considers the purification of omega-3 fatty acids as a group, but does not consider the purification of individual fatty acids. While observing that omega-6 fatty acids, including ARA, are disadvantageous for dietary purposes, the author states that "strains that have less than 1% (as% of total fatty acids) of HUFA C20 can also be isolated: 4n-6 and C22: 5n-6 undesirable for some applications ", which indicates that this author considers that the total amount instead of the relative amount of ARA is important against the present invention. The author does not suggest the method of the present invention, since the method uses a culture of algae without diatoms. Nor do they suggest success in diatomaceous cultures, since the author teaches away from the present invention, depriving the cells of nutrients to increase lipid yield.

No other author has provided methods by which EPA can be obtained in diatoms that have grown heterotrophically in a sufficiently high ratio of EPA to ARA so that it is susceptible to concentration and purification.

In addition, no other author has taught that methods for producing cultured diatoms should comprise avoiding nitrogen limitation and maintaining a minimum concentration of silicate in the culture, preferably by using the prescribed ranges for nutrient component ratios as described. herein, whereby a sufficiently high ratio of EPA to ARA can be obtained.

While methods involving heterotrophic culture of microorganisms, and in particular eukaryotic diatoms of natural origin, can provide an alternative source of omega-3 fatty acids to fish by accumulating large amounts of EPA in the culture, it may seem surprising. that relatively little or no systematic effort has been devoted to the development of such sources of EPA that at the same time are sufficiently free of physiochemically or structurally similar molecules to those of EPA to allow a high degree of concentration and purification. In fact, the current methodology emphasizes the maximization of the absolute amount of EPA and, in doing so, often increases the absolute amount of similar molecules physiochemically or structurally and disproportionately, and therefore hinders the purification of EPA.

Therefore, in a broad aspect, the invention provides a method for obtaining the compositions as described above, wherein the method employs a culture of microorganisms of a type selected for a heterotrophic growth capacity, and an EPA production capacity. , and a relatively low content of molecules physiochemically or structurally similar to EPA; The method includes a culture phase in which cells grow under conditions in which organic carbon is used as an energy source; the conditions preferably include the non-limitation of nutrients selected from a range that includes phosphorus, nitrogen and / or silicon; Such procedures are carried out to maximize the amount of recoverable biomass in which the ratio of EPA to ARA is at least 14: 1 and at least 1.5% w / w of the dry cell weight as eicosapentaenoic acid. However, the inventors have found that although non-limiting levels of phosphate are preferred, the level of phosphate concentration has less effect on the ratio of EPA to ARA and EPA to fatty acids that are concentrated simultaneously compared to the effect of levels. of nitrogen and / or silicate. Non-limiting levels (or greater excesses) of nutrients are very preferred since this may have the effect of substantially increasing the ratio of EPA: ARA in the final product (see Figure 1, for example).

Different modes of culture or fermentation are possible. The simplest of which is batch fermentation in which cells are inoculated in nutrient media, grow over a period of time and then collected. The fed batch fermentation is similar to batch fermentation, but differs in that the concentrated nutrients are supplied to the crop during the growth period. Continuous fermentation involves the continuous harvesting of the crop that includes the biomass and the nutrient solution of the fermentation vessel and its replacement by a fresh nutritive solution. The collection rate in continuous fermentation is chosen so that the density of the cells in culture remains constant. Semi-continuous fermentation is similar to continuous fermentation, except that collections are periodic rather than continuous. Continuous fermentation or semi-continuous fermentation is preferred for the production of the microbial biomass of the invention, but batch fermentation or fed batch fermentation could also be used.

For EPA's industrial production, rates of return are important to provide a cost effective method. Higher production rates are preferred for this reason. Although production rates below 5 mg EPA / L / h may still be useful, the inventors have shown that production rates of more than 5 mg EPA / L / h can be achieved using the methods and processes of the invention where continuous or semi-continuous fermentation / culture techniques are employed. In a preferred option, at least 5 mg of EPA is produced per liter of culture per hour. In a more preferred option, at least 10 mg of EPA is produced per liter of culture per hour.

In addition to the yield rates, the level of EPA (w / w) within the biomass is also important for the industrial applicability of the processes and methods of the invention. In a more preferred option, EPA forms at least 2% of the weight of dry biomass cells, in an even more preferred option EPA forms at least 3% of the weight of dry biomass cells.

In a related aspect, the invention provides a method for producing lipid mixtures and fatty acid mixtures obtained from a culture as previously described in this section, wherein the mixtures are obtained by a collection method that includes the steps of :

collect cells from the culture medium;

optionally, heat or kill cells to denature endogenous enzymes;

form the cells in a biomass cake;

optionally dry the biomass to reduce or remove water;

extract the biomass cake with a non-selective lipid solvent and recover the material extracted from the solvent as a residue; (For example, the use of quasi-critical di-methyl ether is one option, the use of ethanol is another)

optionally treating the extracted material with acids, alkalis and / or enzymes in the presence of an alcohol or water to form mixtures of alkyl esters of fatty acids or fatty acids and optionally concentrating and further purifying said mixtures to achieve a required purity standard.

Biomass can be collected or recovered from the crop according to many methods known to those skilled in the art. These include centrifugation, filtration, sedimentation and decantation. Alternatively, the entire culture can be collected and subjected to drying or lipid extraction procedures.

In a fourth broad aspect, the invention provides a method for culturing diatoms wherein the method employs a culture of diatoms selected for heterotrophic growth capacity and EPA production capacity, and a relatively low content of physiochemically or structurally similar molecules to EPA; The method includes a culture phase in which the cells are grown in a nutrient solution comprising:

a source of organic carbon at an initial concentration of at least 0.5 M carbon;

one or more sources of nitrogen at an initial concentration sufficiently high to ensure that no nitrogen limitation occurs (a mole ratio of organic carbon to nitrogen not exceeding 30 (mol C: mol N) is preferred);

a concentration of bioavailable silicon in amounts sufficient to not limitation silicon occurs (preferably the bioavailable silicon is in the form of silicate and silicate concentration preferably does not fall below 150 | j M).

In a preferred aspect, the initial concentration of the organic carbon source is 0.5 M to 10 M, 1 M to 10 M, or 5 M to 10 M.

In a preferred aspect, the ratio of organic carbon to nitrogen is 1 to 30, 5 to 30, 10 to 30, or 15 to 30.

In a preferred aspect, the mole ratio of organic carbon to phosphorus in the form of phosphate is not greater than 1250 (mol C: mol P), in a more preferred aspect, the mole ratio is not greater than 750. Typically, the mole ratio is 100 to 1250, 250 to 1250, 250 to 750 or 350 to 650 (mol C: mol P).

The bioavailable silicate can take the form of an alkali metal silicate. Preferably sodium metasilicate or potassium metasilicate is used.

The concentration of silicate in the form of silicate is not less than 150jM at any time during cultivation, in a more preferred aspect, the concentration is not less than 200jM. Normally, the lowest concentration is 150jM at 1.3mM, from 200jM to 1mM, from 200jM to 750jM or from 200jM to 500jM. Although the authors have found that it is beneficial for the silicate-free concentration in the crop to remain above 150jM during cultivation, it is likely that small drops below this concentration do not greatly affect the ratio of EPA to ARA obtained. Therefore, slight decreases below 150 µM of the silicate concentration should not be considered as departing from this aspect of the invention. As shown in Table 7 and Figure 1, EPA to ARA ratios can be achieved in the range of 11: 1 to 13: 1 without free silicate in the culture. However, above a concentration of 150 µM, the ratio of EPA to ARA increases significantly. The most preferred maintained concentration of free silicate in the culture is in the range of 200-700jM, more preferred is in the range of 400-600j M. The results shown in Figure 1 and Example 3 show an increase. surprisingly high in the ratio of EPA to ARA at these more preferred concentration ranges. This surprising result was unexpected and indicates the presence of a synergistic interaction between the components.

Most preferably, the nutrient solution comprises an initial concentration of organic carbon source of 0.5 M to 10 M, a mole ratio of organic carbon to nitrogen of 1 to 30, and a concentration of silicate in the form of silicate which does not falls to less than 150 | j M at any time.

In one option, the crop is harvested continuously or discontinuously at an average speed such that, over time, the biomass is removed at the same rate at which it grows.

In a preferred option, the biomass is produced at a rate such that at least 5 mg of EPA is produced per liter of culture per hour. In a more preferred option, at least 10 mg of EPA is produced per liter of culture per hour.

In another preferred option, EPA forms at least 1.5% of the weight of dry biomass cells. In a more preferred option, EPA forms at least 2% of the weight of dry biomass cells, in an even more preferred option EPA forms at least 3% of the weight of dry biomass cells.

In a related aspect, the invention provides a method for producing lipid mixtures and fatty acid mixtures obtained from a culture as previously described in this section, wherein the mixtures are obtained by a collection method that includes the steps of :

collect cells from the culture medium;

optionally, heat or kill cells to denature endogenous enzymes;

form the cells in a biomass cake;

optionally dry the biomass to reduce or remove water;

extract the biomass cake with a non-selective lipid solvent and recover the material extracted from the solvent as a residue (the use of quasi-critical di-methyl ether is an option, ethanol is another option);

optionally treating the extracted material with acids, alkalis and / or enzymes in the presence of an alcohol or water to form mixtures of alkyl esters of fatty acids or fatty acids; Y

optionally, concentrate and purify said mixtures to achieve a required purity standard.

Examples

The following general experimental techniques were used:

Determination of the dry weight of the biomass.

The dry weight of the biomass is measured using a previously weighed fiberglass filter method as follows. A 10 ml sample is extracted from a larger representative sample taken while stirring to achieve a broadly homogeneous dispersion of cells and cell aggregates. The 10 ml sample is placed in a centrifuge tube and rotated at 3000 rpm in a Heraeus Sepatech Megafuge 1.0 with an oscillating rotor for 4 minutes and the liquid is decanted leaving a cellular sediment. The cell pellet is washed with phosphate buffered saline and centrifuged again. A Sartorius fiberglass filter is washed by passing 100 ml of deionized water through the filter and then placing it in an oven at a temperature of 60 ° C for two hours before being weighed. The 10 ml sample is passed through the previously weighed filter in a vacuum filter apparatus, washed with 50 ml of deionized water and then placed in an oven at a temperature of 60 ° C for two hours before returning to to weigh. The difference in grams between the previous and subsequent weights is taken 100 times as a measure of dry weight per liter.

Collection and extraction of lipid material.

The cells are collected and washed to remove excess media. The wet equivalent of 0.75 g of dry cell weight of cellular material is used to produce cell extract. The cell extract containing the lipids can be obtained by Folch extraction following the method of Bligh and Dyer (1959).

Analysis of total fatty acids.

Total fatty acid analysis of cell extract samples are obtained to identify the composition of the cultured material. The addition of an internal standard such as C23: 0 to the reaction allows the measurement of the total fatty acid content of the cells. The method of producing fatty acids involves a basic transesterification with 0.5 M methoxide in methanol, followed by an acid transesterification using dry HCl in methanol. The fatty acid methyl esters are recovered by extraction with hexane and dried with sodium sulfate before analysis using gas chromatography. The sample is processed in a 30 m x 0.25 mm ID Famewax glass (crosslinked polyethylene glycol) capillary column contained in a 2010 GC Shimadzu by self injection. Acids Fatty are identified by comparing peak retention times under standard operating conditions with those of known standards supplied by Sigma Aldrich.

Calculation of nutrient ratios.

To allow comparisons of media with different sources of carbon and nitrogen, nutrient ratios within the media are reduced to molar ratios of constituent atoms, for example, one mole of glucose contains 6 moles of carbon, while 1 mole of acid Acetic contains only 2 mol of carbon.

For the purposes of these calculations, the molecular weight of glucose is 180.16, so that 100 g of glucose contain 6x100 / 180.16 moles of carbon.

For the purposes of these calculations, it is assumed that the yeast extract has a total nitrogen content of at least about 10% (w / w), so that 100 g of yeast extract contains at least about (10/100 ) x100 / 14 moles of nitrogen. Similarly, tryptone has a nitrogen content of at least about 13% (w / w).

For the purposes of these calculations, it is assumed that yeast extract and tryptone contribute an insignificant amount of carbon as a source of energy.

The molar nitrogen content of nitrates and the molar silicon content of silicates can be calculated directly using the molecular weight of each compound.

Example 1

3 g (dry weight) of a strain of Nitzschia laevis In1 was transferred in a stirred tank fermenter with a working volume of 19.4 L. The vessel contained growth media with salts and vitamins as detailed in table 1 together with nutrients in concentrations of: 30 g / L glucose, 1.2 g / L yeast extract, 1.3 g / L sodium nitrate, 40 mg / L potassium dihydrogen phosphate and 270 mg / L sodium metasilicate pentahydrate.

Table 1: Salt and vitamin content of fermenting media.

The culture was aerated with a volume of sterile air container per minute and stirring was controlled to give a dissolved oxygen content of> 50%. The pH was maintained at 8.0 by the addition of 0.4 N NaOH. The temperature was maintained at a temperature of 20 ° C by the circulation of hot or cold water through a jacket around the fermenter vessel, as required.

Attached to the reactor was a settling device designed to separate the media from the cells. The culture was pumped into the device and the cells were returned to the main fermenter vessel, while the spent medium can be removed as waste. Fresh sterile media of the same composition as the initial medium was added to the fermenter vessel to keep the volume of the vessel constant at 19.4 L.

The amount of medium removed as waste was gradually increased during the growth period until 19.4 L of medium was removed during a 24-hour period when the culture density reached the collection density of about 10.5 g / L dry weight.

Once the culture reached harvest density, the culture containing algae cells was purged as collected material at a rate whereby the dry weight of the culture was maintained between 10 and 11 g / L. The volume of spent medium removed as waste was adjusted to keep constant the total volume removed from the reactor per day at 19.4 L. The volume removed from the fermenter was replaced with fresh medium with the same composition as the initial medium.

A steady state was reached where cell density, collection and composition remained stable. The concentration of glucose in the waste medium was greater than 5 g / L, which indicates that the cells were not limited by carbon.

The nutrient content of the fresh medium in the container was modified to contain 2.1 g / L of sodium nitrate and 80 mg / L of potassium dihydrogen phosphate with all other components that remained at their previous levels.

The continuous culture was maintained for a further 48 hours to allow the cells to adjust to the new nutrient levels. During this time, the collection rate required to maintain the same dry weight of the crop increased by approximately 25%, indicating that the crop had a higher growth rate when a higher level of nitrogen and / or phosphate was provided, which indicates that the culture had previously been limited nitrogen and / or phosphate. The concentration of glucose in the waste medium remained above 5 g / L, indicating that the cells were not again limited in carbon.

Sterile samples of biomass were obtained from the cultures both before the change of nutrients and after the change, and lipids were extracted from them. The fatty acid profiles and the proportions of the components within the extract are shown in Table 2 which shows how the change in the culture conditions has resulted in an improvement in the composition with respect to the EPA: ARA ratio and an improvement in the ratio of EPA to components that concentrate simultaneously. EPA formed 2.3% of the dry cell weight after the nutrient change.

Additional increases in the sodium nitrate content of the media after this sample did not produce a marked increase in the growth rate, indicating that the culture was no longer limited by nitrogen.

Table 2: Percentages and ratios of culture fatty acids before and after the change in nutrient conditions. The percentages of fatty acids present are given as a percentage of the total fatty acid content (w / w). Note that, to simplify, fatty acids with 16 or less carbons have not been included in the table where they contribute less than 1% of the total fatty acids. When fatty acids of 18 carbons or more have been omitted from the table, they have not been detected. In particular, juniperonic acid or sciadonic acid could not be detected.

Table 3: Relationships in moles of carbon to nitrogen in limiting and non-limiting nitrogen media.

Example 2

3 g (dry weight) of a culture of a strain of Nitzschia laevis In1 was transferred to a stirred tank fermenter with a working volume of 18 L. The vessel contained growth media with salts and vitamins as detailed in table 1 together with nutrients in concentrations of: 30 g / L glucose, 1.6 g / L yeast extract, 2.98 g / L sodium nitrate, 80 mg / L potassium dihydrogen phosphate and 270 mg / L sodium metasilicate Pentahydrate

The culture was aerated with a volume of sterile air container per minute and stirring was controlled to give a dissolved oxygen content of> 50%. The pH was maintained at 8.0 by the addition of 0.4 N NaOH. The temperature was maintained at a temperature of 20 ° C by the circulation of hot or cold water through a jacket around the fermenter vessel, as required.

Attached to the reactor was a settling device designed to separate the media from the cells. The culture was pumped into the device and the cells were returned to the main fermenter vessel, while the spent medium can be removed as waste. Fresh sterile media were added to the fermenter vessel to keep the volume of the vessel constant at 18 L. The media contained salts according to table 1 together with nutrients in concentrations of: 30 g / L glucose, 1.6 g / L yeast extract , 2.98 g / L sodium nitrate, 80 mg / L of potassium dihydrogen phosphate and 530 mg / L sodium metasilicate pentahydrate. The medium contained a C: N mole ratio of less than 30: 1 (see table 5) to avoid nitrogen limitation.

The amount of medium removed as waste was gradually increased during the growth period until 18 L of medium was removed during a 24-hour period when the crop density reached the crop density of about 10 g / L dry weight.

Once the crop reached crop density, the crop containing algae cells was purged during harvesting at a rate at which the dry crop weight was maintained between 10 and 11 g / L. The volume of spent medium removed as waste was adjusted to keep constant the total volume removed from the reactor per day at 18 L. The volume removed from the fermenter was replaced with fresh medium with the same composition as the initial medium.

A steady state was reached where cell density, collection and composition remained stable. At this point, 6.1 g of dry cell weight were collected per 24 hours per liter of culture. At a silicate requirement of approximately 325 pmol per g dry weight, the daily silicate requirement for biomass synthesis was 1986 pmol L-1 day-1. The silicate supply was 2491 pmol L-1 day-1 giving an Si concentration in excess of 505 pmol. Sterile samples of biomass were obtained from the culture and lipids were extracted from it. The fatty acid profile and the proportions of components within the extract are shown in Table 4. The EPA formed 2% of the dry cell weight under these conditions, which led to an EPA yield rate of EPA 5.1 mg. per liter of culture per hour.

Table 4: Percentage of fatty acids present as a percentage of the total fatty acid content (w / w). Fatty acid ratios and culture percentages during steady state. Note that, to simplify, fatty acids with 16 or less carbons have not been included in the table where they contribute less than 1% of the total fatty acids. Where fatty acids of 18 carbons or more have been omitted from the table have not been detected. N.D. - not detected.

Table 5: Ratio in moles of carbon to nitrogen in the media.

Example 3

3 g (dry weight) of a strain of Nitzschia laevis In1 was transferred in a stirred tank fermenter with a working volume of 14 L. The vessel contained growth media with salts and vitamins as detailed in table 1 together with nutrients in concentrations of: 50 g / L glucose, 1.9 g / L yeast extract, 4.0 g / L sodium nitrate and 170 mg / L potassium dihydrogen phosphate. Sodium metasilicate pentahydrate was added separately as a concentrated stock solution at a concentration of 143 mg / L.

The culture was aerated with a volume of sterile air container per minute and stirring was controlled to give a dissolved oxygen content of> 50%. The pH was maintained at 8.0 by the addition of 0.4 N NaOH. The temperature was maintained at a temperature of 20 ° C by the circulation of hot or cold water through a jacket around the fermenter vessel, as required.

The medium contained a C: N mole ratio of less than 30: 1 (see table 6) to avoid nitrogen limitation. All measurements of the levels in the collected media confirmed that there was> 80 mg / L of nitrate in the culture. Since the fermenter is filled with fresh medium immediately after harvesting, this measure represents the lowest nitrate concentration in the fermenter and, therefore, nitrogen was not limiting throughout this example.

Table 6: Ratio in moles of carbon to nitrogen in the media.

Once the culture reached the collection density, the culture containing algae cells was collected at intervals of six hours at a variable volume, whereby the dry weight of the culture was maintained around 7.5 g / L. The volume removed from the fermenter was replaced with fresh medium with the same content of glucose, sodium nitrate, yeast extract and potassium dihydrogen phosphate as the initial medium.

Silicon as a concentrated aqueous solution of sodium metasilicate was added independently of other media and added at regular intervals throughout the day instead of all at the time of recharging the medium to avoid problems of precipitation at high concentrations. The frequency of addition of sodium metasilicate was modified to vary the concentration of silicon in the culture medium.

Samples were taken for the analysis of fatty acids at daily intervals and the EPA: ARA ratio within the biomass was calculated.

The levels of sodium metasilicate available in the fermenter were calculated by taking the residual silicate levels, adding the amount of silicate added, subtracting the amount incorporated into the biomass (to 325 pmol of silicon per gram of dry weight) and compensating for the dilution caused when the fermenter was collected and filled with silicate free media.

The results shown in Table 7 and Figure 1 show a strong correlation between the available silicate and the EPA: ARA to EPA: ARA residual ratio below 14: 1 in all cases where the silicate levels in the crop were limited (i.e. fell to zero). Surprisingly, a ratio greater than 14: 1 was not observed until the minimum level of sodium metasilicate was above 150 pM.

For all samples where measurements were taken (samples 3 to 11 in table 7), EPA formed at least 3% of the dry weight of the biomass.

Table 7: EPA relationships: ARA in biomass formed in media with different minimum concentrations of sodium metasilicate.

Example 4

The biomass was produced according to the previous method and the lipids were extracted from the lyophilized biomass. The lipids were converted to free fatty acids using KOH in ethanol: water 2: 1, dried to remove water and then converted into ethyl esters using H2SO4 in ethanol.

The resulting ethyl esters were concentrated according to the methods described by WB Nilsson ( "Supercritical Fluid Technology in Oil and Lipid Chemistry", Editors JW King & GR, AOCS Press, ISBN 0-935315-T1-3, 1996, pp. 180 212). Fatty acid concentrations with 20 carbons or more before and after the concentration are shown in Table 8.

Note that, while the relative proportions of each of these fatty acids have increased several times, the ratio of EPA to ARA has only changed slightly, demonstrating the advantage of generating starting material with a high ratio of EPA to ARA.

Table 8: Fatty acids with 20 carbons or more before and after concentration.

Example 5

Mortierella renispora, a fungus of the genus Mortierella, is grown heterotrophically under conditions chosen to produce biomass with a fatty acid composition comprising an EPA to ARA ratio of approximately 11: 1 or more and an EPA to total fatty acid ratio which concentrate simultaneously of approximately 8: 1 or more. Biomass is collected and lipids are extracted. The extracted lipids are transesterified to form ethyl esters. The ethyl esters are then processed to produce a fatty acid ethyl ester concentrate in which the EPA forms at least 60% of the total fatty acids by weight, and this concentrate is then purified to produce an additional EPA ethyl ester. of 90% purity.

Example 6

Chlorella minutissima, an algae of the genus Chlorella, is grown heterotrophically under conditions chosen to produce biomass with a fatty acid composition comprising an EPA to ARA ratio of approximately 11: 1 or more and an EPA to total fatty acid ratio which concentrate simultaneously of approximately 8: 1 or more. Biomass is collected and lipids are extracted. The extracted lipids are transesterified to form ethyl esters. The ethyl esters are then processed to produce a fatty acid ethyl ester concentrate in which the EPA forms at least 60% of the total fatty acids by weight, and this concentrate is then purified to produce an additional EPA ethyl ester. of 90% purity.

Example 7

Nannochloropsis oceanica, an algae of the genus Nannochloropsis, is grown heterotrophically under conditions chosen to produce biomass with a fatty acid composition comprising an EPA to ARA ratio of approximately 11: 1 or more and an EPA to total fatty acid ratio which concentrate simultaneously of approximately 8: 1 or more. Biomass is collected and lipids are extracted. The extracted lipids are transesterified to form ethyl esters. The ethyl esters are then processed to produce a fatty acid ethyl ester concentrate in which the EPA forms at least 60% of the total fatty acids by weight, and this concentrate is then purified to produce an additional EPA ethyl ester. of 90% purity.

Example 8

A screen is carried out after the Barclay method (US 5130242) to identify a Thraustochytrid sp. in which the level of EPA as a percentage of total fatty acids is high in relation to the proportion of ARA and fatty acids that are concentrated simultaneously. The microorganism then grows under heterotrophic conditions chosen to produce biomass with a fatty acid composition comprising a ratio of EPA to ARA of about 11: 1 or more and a ratio of EPA to total fatty acids that simultaneously concentrate of about 8: 1 or more. Biomass is collected and lipids are extracted. The extracted lipids are transesterified to form ethyl esters. The ethyl esters are then processed to produce a fatty acid ethyl ester concentrate in which the EPA forms at least 60% of the total fatty acids by weight, and this concentrate is then purified to produce an additional EPA ethyl ester. of 90% purity.

Example 9

Cyclotella cryptica, a diatom, is cultured heterotrophically under conditions chosen to produce biomass with a fatty acid composition comprising an EPA to ARA ratio of approximately 14: 1 or more. Biomass is collected and lipids are extracted. The extracted lipids are transesterified to form ethyl esters. The ethyl esters are then processed to produce a fatty acid ethyl ester concentrate in which the EPA forms at least 60% of the total fatty acids by weight, and this concentrate is then purified to produce an additional EPA ethyl ester. of 90% purity.

Example 10

Phaeodactylum tricornutum, a diatom that is a mandatory photoautotroph, is transformed according to the method of Apt et al. (US Patent 7939710) to give it the ability to grow heterotrophically using glucose as a source of energy and carbon. The transformed organism is cultured heterotrophically under conditions chosen to produce biomass with a fatty acid composition comprising an EPA to ARA ratio of approximately 14: 1 or more. Biomass is collected and lipids are extracted. The extracted lipids are transesterified to form ethyl esters. The ethyl esters are then processed to produce a concentrate of fatty acid ethyl ester in which the EPA forms at least 60% of the total fatty acids by weight, and this concentrate is then purified to produce an EPA ethyl ester of more than 90% purity.

general

Therefore, the examples show compositions (including compositions of microbial biomass and fatty acids) containing in the region of (or greater than) 20% of e Pa by weight of total fatty acids, with an EPA to ARA ratio of 11: 1 or greater, and with a ratio of EPA to fatty acids that are simultaneously concentrated at 8: 1 or more. It has also been shown that the compositions can be purified to more than 70% EPA by weight of total fatty acids, while at least preserving the ratio of EPA to ARA in the composition.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprises", "comprising", and the like, should be interpreted in an inclusive sense rather than an exclusive or exhaustive sense, that is, in the sense of "that includes, but is not limited to."

Claims (12)

1. A heterotrophically cultured diatom that comprises eicosapentaenoic acid and arachidonic acid in a ratio of at least 17: 1, where eicosapentaenoic acid forms at least 1.5% w / w of the dry weight of the diatom and diatom is Nitzschia laevis . 2. A method for producing a diatom that comprises eicosapentaenoic acid and arachidonic acid in a ratio of at least 14: 1 and at least 1.5% w / w of the weight of dry cells such as eicosapentaenoic acid comprising: to. culturing diatoms in heterotrophic cultures that contain a nutrient solution comprising: (i) an organic carbon source at an initial concentration of at least 0.5 M carbon (ii) a mole ratio of organic carbon to nitrogen of not more than 30: 1 (mol C: mol N) and; (iii) a non-limiting source of silicate that does not fall below 150 pM; Y b. recover the diatom of the heterotrophic culture. 3. The method of claim 2 wherein the diatoms are cultured in heterotrophic culture in a fermenter. 4. The method of claim 3 wherein the culture comprises continuous fermentation. 5. The method of claim 2 wherein the diatoms comprise Nitzschia species . 6. The method of claim 2 wherein said carbon source comprises glucose. 7. The method of claim 2 wherein said carbon source is selected from glucose, hydrolyzed starch or hydrolyzed whey. 8. The method of claim 2 wherein said nitrogen source is in the form of sodium nitrate or potassium nitrate. 9. The method of claim 2 wherein said nitrogen source is enriched with an amino acid source. 10. The method of claim 2 wherein said silicate is in the form of sodium or potassium metasilicate. 11. The method of claim 2 wherein the nutrient solution comprises phosphorus in the form of phosphate. 12. The method of claim 11, wherein the mole ratio of organic carbon to phosphorus in the form of phosphate is not greater than 1250: 1 (mol C: mol P).